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Aims : To understand the basic principle of colorimetry and application of Beers Law for determination of phosphate in various water

r samples. To measure the concentration of phosphate in river , lake and unknown water sample and thus to estimate the fate of phosphate pollution . Introduction : Phosphorus is one of the most important elements essential for growth and maintenance of living organisms in the environment. It is vital and irreplaceable element as the basic building blocks of living cell structure like DNA and RNA contains phosphate in it. Adinosine Triphosphate , one of the form of phosphate is considered as currency of energy transfer in almost all biochemical reactions. To understand the behaviour of phosphorus in environment , it is necessary to consider its chemistry. In fact the natural form of phosphorus is limited to phosphoric acid which can be available as polyphosphoric acids depending upon the polymerization of it. The only stabilized form of phosphorus available in nature is phosphate and as it is non volatile ,thus it is restricted to lithosphere and hydrosphere only. This fully oxidized form has a formal oxidation number V and co-ordination number 4. In almost all minerals it is available as ortho phosphate , which can be represented as PO4-3 . This phosphate is quite mobile and it has a tendency of moving from lithosphere to hydrosphere . It has a tendency to concentrate in such places where it is almost not available for recycling such as bottoms of lakes and sea. This characteristics movement of phosphate creates a shortage of this nutrient making it growth limiting for ecological systems. One of the most important properties of phosphate and polyphosphate is to form chelates , complexes and insoluble salts with metal ions in water environment. It is dependent upon the concentration of phosphate and metal ions , pH and presence of other legands in water. In fact this complexion and chelation greatly affects the distribution of phosphate in aquatic environment as its concentration is very low compared to metal ions. Phosphate is found in the dissolved form in natural waters as a result of the natural weathering and solution of phosphate minerals, soil erosion and transport , soil fertilization and resultant phosphorus transport , biological transfer and use of soluble phosphate compounds in detergent manufacturing , water treatment and industry. Many chemical . physical and biological factors are significant in describing the phosphorus cycle. It can be divided in two biological cycles in water and soil and one inorganic cycle. In primary inorganic cycle phosphate enters from the ingeneous rock and transfers through soils and various water bodies like sea , lakes and rivers where it is ended up as sediment. Depending upon the type of water bodies the chances of phosphorus

ended up as sediment are different. In case of deep sea there is little chance of returning the phosphorus settled in sediments back to the cycle. While in case of lake some amount of phosphorus is returned back to the cycle . In water based biocycle of phosphate bacteria , algae and various carnivores surviving on them are the key carriers of phosphate within the aquatic environment. In fact the extent of biomass in most lakes and sea is governed by phosphate concentration and available solar energy. Algae which is analogous to land plants in aquatic systems can take up more amount of phosphate than they need and store the surplus amount to support cell divisions without outside help. In fact they are considered as producer organisms which convert in organic nutrients in to complex molecules which help biomass to grow in aquatic systems.Due to excess supply of phosphate received from rivers passing through cities , the algal bloom and eutrophication is a common phenomenon in many lakes across the globe. Algae can take up both organic and inorganic phosphate in them and release the phosphate rapidly upon dying. Phosphate will stimulate the growth of plankton and aquatic plants which provide food for larger organisms, including: zooplankton, fish, other mammals.Thus phosphate in water has a net downward movement due to the debris and faecal of these carnivores and algae .Finally this phosphate reaches to the bottom of the lake and remain there for millions of years. There are some chances of returning the phosphate from the top layer of sediments due to activities of decomposer organisms, enzymes and worms. The stratification of lakes which creates reduced oxidation and acidic state pulls out phosphate by converting insoluble forms of FePO4 and CaHPO4 to soluble Fe3(PO4)2 and Ca(H2PO4)2 respectively. Still the overall mechanism is such that phosphate is lost from the cycle creating a shortage of it. As the overall concentration and cycling of phosphate is very less in environment , the anthropogenic activities like use of phosphate fertilizers and phosphate based detergents has triggered the pollution of receiving water bodies due to phosphate. Following Eutrophication of some great lakes in North America , a detergent phosphate ban was employed in 1970s in USA and Canada. The addition of tertiary advanced treatment to remove phosphate in wastewater treatment is also the impact of phosphate pollution caused due to anthropogenic activities. The criteria for phosphate concentration in water bodies are as follows : Designated Use Limit Freshwater Aesthetics Federal Criteria: (USEPA, 1986) --streams/rivers 0.1 mg/l --streams entering lakes 0.05 mg/l --lakes/reservoirs 0.025 mg/l (Ref:http://webpages.charter.net/kwingerden/erhs/aquarium/phosphat.htm#CriteriaFor Phosphorus)

Phosphorus occurring as orthophosphate can be measured quantitively by gravimetric, volumetric or colorimetric methods. The gravimetric method is applied for higher concentration which is rarely in water.Volumetric methods can measure up to 50 mg/lit of phosphate but involves precipitation, filtration and careful washing and titration of the precipitate which is time consuming and thus not much popular . The colorimetric and spectrophotometric methods due to their speed , simplicity and accuracy for detecting even a small concentration in the range of micro gram per litre are quite popular in measuring phosphate and other nutrients in water. In colorimetric analysis the constituent for determination in water is reacted with a reagent to form coloured complex and with suitable visual or instrumental colorimetric method the intensity of the coloured complex is measured to get the concentration of the constituent in water. Whenever a light with intensity (Io) is passed through the coloured complex , it will absorb the light corresponds to the colour which in turn proportionate to the concentration of constituent in sample, thus it is imperative to establish a relationship between the intensity of light absorbed and concentration of constituent under measurement. There are two laws which establish this relation. (i) Lamberts Law : It states that the amount of light absorbed by the coloured complex is directly proportional to the length of the light path, i.e the length of the coloured complex. If Io= initial intensity of light and I= intensity of light transmitted and l = length of sample , then T = I / Io = 10 kl ------------------- (1)

The term (I/Io) is called the transmission and it is the amount of light transmitted through the sample. Taking logarithm , Log (Io/I) = -kl = A --------------------- (2)

The term log(Io/I) represent the amount of light absorbed by the sample and it is called absorbance. (ii) Beers Law : It states that the amount of absorbance increases as the concentration of coloured complex is increased.

Therefore , Log(I/Io) = -Kc ----------------- (3) where c = concentration . Thus , Absorbance A = e c l ------------------- (4) Where e is called molar absorpitivity and it is personal characteristics of constituent under measurement . It represent the absorbance of 1M solution in 1 cm cell. It also depends upon the wavelength of light passed through the coloured complex. To increase the sensitivity of the test , a wavelength for absorbance is selected which has maximum molar absorptivity. In case of blue coloured complex used in phosphate measurement , this wavelength corresponds to 880 nm and thus the test is conducted at this wavelength.

From equation (4) it is apparent that for a wavelength corresponds to maximum molar abosptivity and fixed length of light path , the absorption of light by coloured complex is directly proportional to the concentration of constituent in the solution. The length of the light path is fixed by using a standard size of cuvette for measurement of absorbance in the colorimeter.Thus measuring the absorbance for standard solutions , a graph of absorbance against concentration is obtained which is straight line for all the measurement which follow Lambert and Beers Law as mentioned above . The concentration of unknown sample can be obtained by measuring the absorbance and then plotting the value on the calibration curve the concentration of unknown sample is also obtained. The colorimetric methods are applicable to surface waters , domestic and industrial waste waters which have orthophosphate concentration in the range of 3 micro gram /lit to 100 micro gram /lit. In this method ammonium molybdate and potassium antimonyl tartrate react in an acid medium with dilute solutions of phosphate to form an antimony-phospho-molybdate complex. The complex is reduced to intensely blue coloured complex by ascorbic acid . The colour is proportional to the phosphorus concentration and that is measured on 880 nm wave length . Methodology : From a standard solution containing 2.5 mg/lit phosphate , a series of diluted solutions having concentrations of 0.125 mg/lit , 0.250 mg/lit , 0.500 mg/lit and 1.000 mg/lit phosphate were prepared in conical flasks. A 50 ml sample was taken from each concentration solution and 8 ml of combined reagent was added to each sample. The combined reagent contained in it 2.5M Sulphuric acid , potassium antimonyl tartrate , ammonium molybdate and ascorbic acid in it. The solution was allowed to stand for 10 to 30 minutes. During this period due to the reaction of potassium antimonyl tartrate and ammonium molybdate in the presence of sulphuric acid developed antimony-phospho-molybdate complex which was converted to blue coloured complex in the presence of ascorbic acid . The river , lake and unknown water samples were treated in the same way and by adding the combined reagent to each sample blue coloured complex was formed. Then filling the sample cells with the solution and a blank filled with distilled water the spectrophotometer was set to zero absorbance reading. Putting the blank sample in the spectrophotometer holder and putting the samples one by one the absorbance for each sample was measured. For , standards solution the absorbance against concentration graph was prepared and from this calibration graph the concentration of phosphate for river , lake and unknown water sample was obtained. As to obtain a calibration curve which is close to Beers Law , it is necessary that all the results of the standards solution absorbance fall on the line of the graph. It mean all the points must have a linear relationship. To obtain this linear relation ship it is necessary to dilute the solutions further for which absorbance values are too high . Thus the river water sample was further diluted to obtain a linearity for the calibration curve.

Results and Calculations : The absorbance reading for all the groups for different samples are summarised in the table given below : Group 1 2 3 4 5 6 7 0.125 mg/lit 14 14 6 14 5 12 5 0.250 mg/lit 30 17 12 25 8 27 13 Absorbance Reading 0.500 1.0 Lake mg/lit mg/lit 44 33 16 27 49 12 26 50 12 41 87 13 29 68 10 51 103 11 20 42 10 River 44 (1:1) 37 88 69 86 102 62 Unknown 24 23 18 18 10 19 18

Table 1 : Group Absorbance values for standard solutions and samples. From the above table the calibration curve for absorbance against concentration can be plotted and from the graph for different values of absorbance for samples the phosphate concentration of river , lake and unknown water sample can be obtained. For river water the sample was dilute for two time with (1:1) dilution ratio. Thus from the graph the concentration obtained was 0.39 mg/lit which was for diluted solution. Thus the concentration of phosphate in river water was 0.39 x 2 = 0.78 mg/lit. The concentration of phosphate obtained from the calibration curve are summarised in the following table. Phosphate Concentration (mg/lit) Group Lake Water River Water Unknown Water 1 0.14 0.78 0.21 2 0.1 1.45 0.375 3 0.237 0.787 0.246 4 0.1125 0.787 0.169 5 0.19 0.81 0.19 6 0.1125 0.9875 0.1875 Table 2 : Phosphate Concentration in the Water Samples Statistics : The descriptive statistics for the class results of phosphate are summarised as follows :
Descriptive Statistics: Phosphate
Variable Phosphate Water Source L R U Mean 0.1487 0.934 0.2296 Maximum 0.2370 1.450 0.3750 SE Mean 0.0220 0.108 0.0310 StDev 0.0540 0.265 0.0759 Minimum 0.1000 0.780 0.1690 Q1 0.1094 0.785 0.1829 Median 0.1263 0.799 0.2000 Q3 0.2018 1.103 0.2783

Variable Phosphate

Water Source L R U

Note : L = Lake Water , R = River Water , U = Unknown Water .

The box plot of the phosphate concentration for all the samples are given as below :
Boxplot of Phosphate vs Water Source
1.6 1.4 1.2 Popa h s h te 1.0 0.8 0.6 0.4 0.2 0.0 L R Water Source U

Looking at the box plot of the phosphate concentration of all the samples , it is evident that the spread of data is different in each sample as the sources of the sample are different and the concentration of the phosphate varies in the sample according to the source. The location of median line and mean symbol for each sample suggests that the average concentration of phosphate in lake sample is the lowest while that of river is highest of all. The summary statistics confirms the impression with a mean value of 0.1487 mg/lit for lake water and 0.934 mg/lit for river water with a median of 0.1263 mg/lit and 0.799 mg/lit respectively. The size of interquartile range boxes in lake water and unknown sample indicates that the spread of data is narrowed over a particular range . While the size of interquartile range box in river water indicates that the data is quite spreaded across the values . In fact in all three samples the data is positively skewed which may be the possible effect of maximum values in each sample.
Correlation and Linear Regression analysis : Correlations: concentration, absorbance
Pearson correlation of concentration and absorbance = 0.523 P-Value = 0.477

The regression equation for absorbance and concentration can be summarised as follows . The detail regression analysis is given in the appendix 1. Concentration = (SE) -0.026 (0.6038) + 0.0163 absorbance

( 0.01881)

N=4 , R2 = 27.4 % , S = 0.403804 .

The value of R-square suggest a poor fit for linear regression and thus indicates a possible deviation from Beers law which suggests that the absorbance is directly proportional to the concentration. Discussion : For Group 1 the values of phosphate concentration in river water is 0.14 mg/lit , for lake water it is 0.78 mg/lit and for unknown sample the value is 0.21 mg/lit. The mean Of river water phosphate concentration is 0.934 mg/lit with standard deviation of 0.265 mg/lit. The mean of lake water sample is 0.1487 mg/lit with a standards deviation of 0.0540 mg/lit while for unknown sample mean is 0.2296 mg/lit and standard deviation is 0.0759 mg/lit. The variations in the results can be accounted to instrumental errors , manual errors and deviation from following the Beers Law . The absorbance of the coloured complex is dependent upon the concentration of phosphate in the sample. Thus manual errors of pipetting the correct amount water sample and reagent may affect the absorbance of coloured complex which in turn may lead towards error in the estimation of concentration of phosphate in the sample. The fundamental assumption of Beers law is that the light is monochromatic and strictly monochromatic light is practically impossible to achieve . In fact the real photometers utilize a band of wave length centring over a particular wavelength. Thus the linearity assumption between the absorbance and concentration can not be followed accurately . The correlation test and simple linear regression of absorbance against concentration for group 1 reveals this fact in a very well manner giving a Rsquare value of 27.4% . Even the calibration curve shows curvature for higher concentration reading which is shown by dotted line in the calibration curve. As the position of chemical equilibrium depends upon the concentration of particular species in the sample , the difference in concentration of phosphate in sample may also cause error in the formation of final coloured complex and thus may lead towards errors in the results. Finally , as the probability of getting a best fit line in calibration cure is infinite , there may be chances of getting different calibration curves among the groups and thus the concentration of water samples. Considering the US EPA criteria for phosphate in water bodies , it is apparent that the phosphate concentration of all the samples is very high and may lead towards algal blooms problem. The waters require treatment for phosphate prior to utilise for drinking water purpose. The possible causes of high concentration of phosphate in river water sample might be anthropogenic activities like agricultural application and use of phosphate based detergent also. If the river is receiving a treated effluent from treatment plant then there is a need for providing a phosphate removal unit process at the treatment plant for removal of phosphate .

Conclusion : The values of phosphate for various water samples are as follows :
Variable Phosphate_L Phosphate_R Phosphate_U N 6 6 6 Mean 0.148667 0.933583 0.229583 StDev 0.053983 0.265114 0.075904 SE Mean 0.022038 0.108232 0.030988 95% (0.092015, (0.655363, (0.149927, CI 0.205318) 1.211803) 0.309240)

The concentration of phosphate in river water and lake sample is very high and may lead towards possibility of algal blooms . Using a better filter to get monochromatic wavelength the errors caused due to a range of wavelengths in measurement of absorbance can be minimized and a better linearity can be achieved for a particular calibration curve. Moreover with smaller range of concentration of standards solutions a better calibration curve can be achieved as the linearity relation between absorbance and concentration is better for smaller concentrations of standards solutions. With careful manual techniques of sampling the variations in the concentrations in different samples can be minimized and a better chemical equilibrium of coloured complex can be achieved which can further help in getting a better calibration curve.

References : 1) Clair N Sawyer , Perry L McCarty and Gene F Parkin , 1994 , Chemistry for Environmental Engineering , McGraw Hill International Editions , 2) D Barnes and F Wilson , 1983 , Chemistry and Unit operations in Water Treatment , Applied Science Publishers . 3) H A Flaschka , A J Barnard Jr and P E Sturrock , 1969 , Quantitative Analytical Chemistry :Vol 1 , Barnes and Noble Inc. 4) John H Kennedy , 1990 , Analytical Chemistry : Principles , Second Edition , W B Saunders Company. 5) Jon C Van Loon , Chemical analysis of Inorganic constituents of Water , 1982 , CRC Press Inc. 6) O.Hutzinger, Edited , 1980 , The Hand Book of Environmental Chemistry : volume 1 Part A , The Natural Environment and the biogeochemical cycles , Springer Verlag Berlin Heidelberg New York . 7) Werner Stumm and James J Morgan , 1970 , Aquatic Chemistry : An introduction emphasizing Chemical Equilibria in Natural Waters , John Wiley & Sons Inc. 8 ) http://webpages.charter.net/kwingerden/erhs/aquarium/phosphat.htm#CriteriaForPhos phorus) Appendix 1 : Linear Regression Test statistics for absorbance and concentration produced in Minitab-14.

Appendix 1 Linear Regression Statistics of Absorbance concentration .


Correlations: absorbance, concentration
Pearson correlation of absorbance and concentration = 0.523 P-Value = 0.477

Regression Analysis: concentration versus absorbance


The regression equation is concentration = - 0.026 + 0.0163 absorbance Predictor Constant absorbance S = 0.403804 Coef -0.0257 0.01635 SE Coef 0.6038 0.01881 T -0.04 0.87 P 0.970 0.477

R-Sq = 27.4%

R-Sq(adj) = 0.0%

Analysis of Variance Source Regression Residual Error Total DF 1 2 3 SS 0.1231 0.3261 0.4492 MS 0.1231 0.1631 F 0.75 P 0.477

Residual Plots for concentration


Normal Probability Plot of the Residuals
99 90 Residual Percent 50 10 1 -0.2 -0.8 -0.4 0.0 Residual 0.4 0.8 0.2 0.4 Fitted Value 0.6 0.4 0.2 0.0

Residuals Versus the Fitted Values

Histogram of the Residuals


2.0

Residuals Versus the Order of the Data


0.4

Frequency

Residual -0.2 -0.1 0.0 0.1 0.2 Residual 0.3 0.4 0.5

1.5 1.0 0.5 0.0

0.2 0.0 -0.2 1 2 3 Observation Order 4